JP3266766B2 - Hepatic parenchymal cells having loan proliferating ability, method for obtaining the same, and method for subculturing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a hepatic parenchymal cell having clonal proliferation ability, a method for obtaining the same, and a method for subculturing the hepatic parenchymal cells. More specifically, the present invention is used as a material for cell biology and molecular biology research on the development / differentiation and division / proliferation processes of hepatocytes, or its canceration mechanism, or for the development of therapeutic techniques for various liver diseases. And a method for obtaining the liver parenchymal cells, and a method for subculturing the liver parenchymal cells.

[0002]

2. Description of the Related Art An animal individual is a multicellular organism in which one fertilized egg repeats division and differentiates into various tissues (cell aggregates) sharing different functions. In the case of each tissue constituting the body, each cell constantly divides and proliferates, and the individual is maintained by actively producing cells capable of expressing a differentiation function one after another. Therefore, in order to understand the biological entity of human and other animal individuals, or to elucidate the mechanisms of carcinogenesis and develop therapeutic methods, the cells that make up each tissue must be analyzed by cell biology and molecular biology. It is important to analyze in detail biologically and to clarify the development / differentiation process and the mechanism of division and proliferation.

Conventionally, as a means for analyzing cells of a living tissue in detail, a method has been established in which cells taken out of a living body are cultured, and further, the cultured cells are divided and proliferated to survive in a subculture. I have. However, for rat and human hepatocytes, it has been considered that it is impossible to subculture primary cells isolated from mature individuals. That is, the adhesion-dependent mature hepatocytes are greatly damaged when detached from the culture substrate due to the subculture operation, and it is difficult to reattach to the culture substrate. It was impossible to study the developmental process of hepatocytes and the state of mitotic proliferation. The inventors of the present invention have overcome the above difficulties by devising the components and the like of the culture medium, succeeded in subculturing primary cells collected from the liver of an adult rat, and have already developed the culture method. A patent application has been filed (Japanese Patent Application No. 6-89056).

[0004] However, in order to understand the complex and diverse functions of hepatocytes or to elucidate the mechanism of their carcinogenesis, pure hepatic progenitor cells (prases with unspecified differentiation directions) have been developed.
It is considered essential to identify ogenitor cells), but its existence has not been confirmed so far, and a selective culture method has not been established. Regarding such hepatic progenitor cells, the following facts have been known so far, and the following attempts have been reported to identify them.
That is, it has been reported that during liver development, stem cells are generated from liver primordium and these stem cells differentiate into hepatocytes and bile duct epithelial cells (Shojiri et al .: Cancer Research, vo.
l.51, pp2611-2620, 1991). This stem cell has not been confirmed in the liver of a mature individual (rat, etc.), but oval cells appearing in the pre-cancerous state of rat liver carcinogenesis can be both liver cancer and cholangiocarcinoma From these facts, it is considered that these oval cells are caused by abnormal differentiation of stem cells present in the liver of adult rats. In addition, Hixson et al. (Pathobiolog
y, vol. 58, pp65-77, 1990) prepared several antibodies against the surface antigen of rat oval cells obtained by a hepatocarcinogenesis experiment.
d., vol. 204, pp. 261-269, 1993) selects cells that bind to these antibodies from adult rat hepatocytes using a cell sorter and investigates their properties. As a result, when these cells were cultured on a culture substrate supplemented with additional factors or on a feeder layer of fetal mesenchymal cells, cells that proliferated or differentiated into mature hepatocytes were observed. This suggested that hepatocyte progenitor cells were included in the cells binding to the antibody against the surface antigen of the elliptic cells.

As described above, the existence of hepatic progenitor cells has been presumed based on several pieces of evidence, but its existence has not been confirmed so far, and hepatic parenchymal cells which are indispensable for identifying progenitor cells. No clonal expansion has been reported. The present invention has been made in view of the circumstances described above, and has as its object to provide hepatic parenchymal cells having clonal proliferative ability, which are considered to contain hepatic progenitor cells. Another object of the present invention is to provide a method for obtaining those cells and a method for continuously culturing those cells.

[0006]

Means for Solving the Problems The present invention first solves the above-mentioned problems by providing the following cell biological characteristics, (1) having peroxisomes, (2) being positive for a hepatocyte marker. At least one of (3) that a part of the colonies is positive for a cancerous or undifferentiated hepatocyte marker, and (4) that it is positive for an antibody against the oval cell surface antigen. A hepatic parenchymal cell having clonal proliferation ability,

Further, the present invention provides a method for separating hepatocytes from the liver of an adult mammal, separating the hepatocytes into a heavy fraction and a light fraction by low-speed centrifugation, and separating the small hepatocytes in the light fraction. Culture in a medium containing bovine fetal serum and ascorbic acids as essential components to form colonies of hepatic parenchymal cells belonging to small cells, or the presence of peroxisomes, reactivity to hepatocyte markers, cancerous liver A liver having a clonal proliferative ability characterized by screening using at least one of reactivity to a cell marker, reactivity to an undifferentiated hepatocyte marker, and reactivity to an antibody to an oval cell surface antigen as an index Provided is a method for obtaining a parenchymal cell.

Further, the present invention provides a method for separating hepatocytes from the liver of an adult mammal, separating the hepatocytes into a heavy fraction and a light fraction by low-speed centrifugation, and separating the small hepatocytes in the light fraction. Primary culture was performed in a medium containing bovine fetal serum and ascorbic acids as essential components to form colonies in hepatic parenchymal cells belonging to small cells, the colony cells were detached from the medium with an EDTA solution, and the detached cells were transferred to the primary culture medium. Re-cultured in a medium consisting of the same composition as, or
The colony cells formed by the primary culture described above were
The cells were detached from the medium with a TA solution and a trypsin solution, and the detached cells were dispersed in individual cells.
The present invention provides a method for subculturing hepatic parenchymal cells having clonal proliferation ability, which comprises reculturing in the medium itself used in the initial stage of the primary culture.

[0009] In the above-mentioned method for obtaining hepatic parenchymal cells and its subculture method, small hepatocytes in the light-weight fraction obtained by low-speed centrifugation are separated from fetal bovine serum, ascorbic acids, epidermal growth factor, Nicotinamides and DM
In a preferred embodiment, the cells are cultured in a DMEM medium containing SO.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION First, the method for obtaining hepatic parenchymal cells having clonal proliferation ability of the present invention will be described in more detail. Usually, in order to collect hepatocytes, a heavy fraction is obtained by low-speed centrifugation (50 G). In the method of the present invention, a light fraction obtained by low-speed centrifugation is separated, and cells contained in this fraction are cultured. I do. The culture medium contains fetal bovine serum (FBS) and ascorbic acids (for example, L-ascorbic acid phosphate). As is clear from the test results of the examples described later, these components cause colonization of small hepatocytes in the non-parenchymal cell fraction.
Also, epidermal growth factor (EGF) and DMSO
Although not essential for colony formation, it has a colony formation-promoting action, and nicotinamides are considered to suppress hepatocyte differentiation, and are therefore preferred as components added to the culture medium. Furthermore, the non-parenchymal cell fraction contains endothelial cells, Kupffer cells, stellate cells, bile duct epithelial cells, etc., in addition to small hepatocytes, and is considered to provide a special environment for small hepatocytes. However, the above-mentioned nicotinamides, ascorbic acids, and DMSO suppress the growth of these non-parenchymal cells, and allow small hepatocytes to be selectively cultured and grown.

The amounts of these components added to the medium are, for example, 5-30% for FBS and 0.1-1.% For ascorbic acids.
0 mM, EGF can be 1-100 ng / ml, nicotinamides can be 1-20 mM, and DMSO can be about 0.1-2%. Culture was performed under 5% CO ₂ conditions for 3 hours.
This is performed at a temperature of about 7 ° C. By the culture as described above, colonies of small hepatocytes that grow clonally are obtained. Furthermore, for cells forming these colonies, for example, the presence or absence of peroxisomes, reactivity to hepatocyte markers, reactivity to cancerous hepatocyte markers, reactivity to undifferentiated hepatocyte markers, and By performing screening using at least one of the reactivity of the oval cell with the antibody against the surface antigen as an index, the expression of a differentiation function as a hepatocyte can be confirmed. Of these, the presence or absence of peroxisomes can be confirmed by observation with a transmission electron microscope. Albumin and α ₁ as hepatocyte markers
-Antibodies such as antitrypsin and transferrin are used as markers for cancerous or undifferentiated hepatocytes as GST-
Antibodies to P, α-fetoprotein, γ-GTP staining, etc., and antibodies (OC2, OC3) prepared by Hixson et al. Can be used as antibodies against the surface antigen of oval cell. Furthermore, non-parenchymal cells in culture can be identified by using a marker for bile duct epithelial cells, a marker for stellate cells, and the like.

Next, the method for subculturing hepatic parenchymal cells having clonal proliferation ability of the present invention will be described. That is, in this method, the colonies of liver parenchymal cells (primary cultured cells) obtained by the above-mentioned obtaining method are peeled off from the Petri dish,
This is a method of reculturing and growing in another Petri dish. When detaching the colony, after removing the medium from the Petri dish, add a 0.002-0.2% EDTA solution and treat at about 37 ° C. for about 10 minutes,
Non-parenchymal cells around the colony and small hepatocytes can be detached from each colony. This is sown in a petri dish, and cultured in the same medium used for the primary culture (that is, a medium containing bovine fetal serum and ascorbic acids as essential components), thereby allowing the colonies and non-parenchymal cells to adhere to the petri dish and growing again. Can be done.

Alternatively, after removing the medium from the petri dish, a solution of EDTA (0.002 to 0.2%) and trypsin (0.005 to 0.5%) is added to the colonies and treated for about 10 minutes. Thus, colonies can be separated into small hepatocytes and non-parenchymal cells. The cells can be individually dispersed by filtering these cell separation solutions with a filter (pore size: about 20 μm) to remove small aggregations. The small cells dispersed in this way are particularly suitable for the medium (condit) used in the initial stage of the primary culture (eg, 1 to 4 days of culture).
By reculturing with an ionized medium), the cells can be divided and proliferated in a favorable state.

The method of the present invention as described above can be applied to hepatocytes of all mammals including humans, and obtains hepatic parenchymal cells having clonal proliferative ability from various animal species. , And their hepatic parenchymal cells can be subcultured. For example, hepatic parenchymal cells capable of clonal proliferation, or subcultured cells thereof, collected from human liver can be used for producing hybrid livers and the like, and are being developed for the development of therapeutic techniques for liver diseases. It is expected to bring.

Hereinafter, the method for obtaining cells of the present invention will be described in more detail and concretely with reference to Examples, and the test results will also be shown for the clonal proliferating hepatocytes obtained by this method. The characteristics and the like will be described. Of course, the present invention is not limited to the following examples.

[0016]

【Example】

Example 1 Liver parenchymal cells of the present invention were obtained by the following method and tested for their cell biological properties. (1) Culture of hepatocytes Liver cells were collected from 4-344 to 22-week-old F344 male rats by collagenase perfusion and centrifuged at low speed (50 g,
The supernatant obtained by 1 minute x 3 times) was further centrifuged at a low speed (150 minutes).
g, 5 minutes x 3 times) to obtain a non-parenchymal cell fraction as a precipitate. The cells were placed in a 3.5 cm diameter culture dish at 9 × 10
Seed ⁵ cells at a time, and in DMEM medium (10% FBS, 44 mM NaHCO ₃ , 2
0 mM HEPES, 0.5 mg / l insulin, 10 ^-7 M dexamethasone, 30 mg / l L-proline, penicillin and streptomycin) in, 37 ° C.,. 2 to under 5% CO ₂
The cells were cultured for 3 hours. Next, the culture medium was added to the above medium for 10 m.
Replaced with DMEM medium supplemented with M nicotinamide, 10 ng / ml EGF and 0.2 mM L-ascorbic acid phosphate,
From the fourth day, 1% DMSO was added to the medium, and the culture was continued. (2) Test method As an indicator of hepatocyte proliferation, BrdU incorporation and photographs of the same site were taken with time under a phase-contrast microscope.
The area of the hepatocyte area was measured.

The expression of hepatocyte function and the identification of non-parenchymal hepatocytes were performed using the oval cell antibodies (OC2 and OC3) and the bile duct epithelial cell marker (BD)
1: Saccharatin 7), hepatocyte marker (albumin, α ₁ -antitrypsin, transferrin antibody), cancerous or undifferentiated hepatocyte marker (GST-P, α-fetoprotein antibody) ,
γ-GTP staining) and immunocytochemical or enzymatic chemical techniques using stellate cell markers (desmin antibodies). In addition, the organelles were observed in detail using a transmission electron microscope.

In order to compare the difference in the culture results of the rats from which hepatocytes were collected, the samples on day 10 of the culture were subjected to HE staining, and the formation rate of hepatocyte colonies was measured under an optical microscope. In addition, the population of 8 or more cells was counted as a colony. The same culture was performed in a system in which FBS, nicotinamide, EGF, L-ascorbic acid phosphate, and DMSO, which were added factors to the culture medium, were removed one by one. The effect of was investigated. (3) Test Results As a result of culturing under the conditions of the above (1), it was observed that small hepatocytes formed colonies and grew clonally, as shown in the phase contrast micrographs of FIGS. These figures 1 to
3 is a phase contrast image (magnification 147) of the same visual field of the cell culture collected from an 8-week-old rat, on the third day of the culture (FIG. 1).
Thus, one small hepatocyte became four populations on day 5 (FIG. 2) and formed about 300 colonies on day 15 (FIG. 3).

As shown in FIG. 4, the appearance rate of colonies decreased as the age of the rat from which hepatocytes were collected was increased. In addition, as shown in FIG. 5, hepatocyte proliferation curves were almost the same regardless of the age of the rat. These results suggested that the hepatocytes that formed the colonies were immature progenitor cells. Further, from the result of HE staining, the colony on the 10th day of culture was composed of uniform small hepatocytes (FIG. 6). A multilayer structure was observed in the portion (FIG. 7). In addition, a positive site for the bile duct epithelial cell marker BD1 (FIG. 8) and a positive site for cytokeratin 7 (FIG. 9) were observed in a part of the hepatocyte colony. From these results, it is considered that the cultured cell colony is a population containing stem cells that differentiate into hepatocytes and biliary epithelial cells.

Next, this cell colony was positive for the hepatocyte marker, and it was confirmed that the cell colony expressed a normal function. That is, FIG. 10 is a micrograph of a colony stained with albumin, and FIG. 11 is a micrograph of a colony stained with α ₁ -antitrypsin.
In addition, from the result of double staining of transferrin and BrdU, BrdU incorporation was observed in transferrin-positive cells as shown in FIG. Furthermore, the cells of this colony are partially positive for cancerous or undifferentiated hepatocyte markers (FIG. 13: α-fetoprotein staining, FIG. 14: GST-P staining, FIG. 15: γ-GTP staining). And oval cell antibodies (FIG. 16: OC2 staining, FIG. 17: OC3 staining).

On the other hand, most of the non-parenchymal cells around the colony were positive for the desmin antibody which is a stellate cell marker, but some cells were negative (FIG. 18). It was confirmed that many other non-parenchymal cells existed. Table 1 shows the results of a test in which the factors added to the medium were removed one by one. In this test, the culture dish on the 31st day of culture was stained with albumin, and albumin-positive cells 8
Table 1 shows the average value (n = 3) of the number of hepatocyte colonies per 1 cm ² with one or more groups as one colony. 19 to 24 show phase contrast micrographs of the colony of each of the system (control) to which all the additional factors were added and the colony excluding each factor.

[0022]

[Table 1]

As is clear from Table 1, in the system without EGF, there was no difference in the growth of non-parenchymal cells as compared with the control, but the formation of hepatocyte colonies was clearly suppressed (FIG. 1).
9 and FIG. 20). On the other hand, in the nicotinamide (-) system, the growth of non-parenchymal cells was promoted, but the formation rate of hepatocyte colonies was not affected. However, in this nicotinamide (-) system, hepatocytes showed a large and cord-like structure, and showed a form that more expresses the differentiation trait of hepatocytes than the control (FIG. 21). In the system of L-ascorbic acid phosphate (-), proliferation of non-parenchymal cells was promoted, but almost no hepatocyte colonies were formed (FIG. 22).
Non-parenchymal cell growth was promoted in the SO (-) system,
The hepatocyte colony formation rate was lower than the control (FIG. 2).
3). In addition, in the case of the FBS (−) system, neither non-parenchymal cells nor hepatocytes could be maintained (FIG. 24).
From these results, it was confirmed that the addition of FBS and ascorbic acids to the culture medium for obtaining the hepatic parenchymal cells of the present invention was essential. In addition, although EGF and DMSO are not essential for the formation of hepatocyte colonies, they have a hepatocyte colony formation promoting action, and nicotinamide was considered to be a factor involved in hepatocyte differentiation. Additionally, nicotinamide, ascorbic acid and D
It has also been found that MSO has a growth inhibitory effect on non-parenchymal cells.

Finally, from observation with a transmission electron microscope, peroxisomes characteristic of hepatocytes were observed in the cytoplasm of the cells constituting the colonies (FIG. 25-a,
b). Example 2 The hepatocytes obtained in Example 1 were subcultured by the following method. The medium was removed from the Petri dish in which the small hepatocyte colonies were formed, and about 10% using 0.02% EDTA.
The colony was peeled off from the petri dish by treating for 1 minute. Next, a Petri dish was filled with a medium having the same composition as that used in the primary culture for colony formation, and the detached colonies were adhered to the Petri dish. Non-parenchymal cells and small hepatocytes around the colony both adhere to the petri dish,
It was confirmed that the cells were well divided and proliferated (FIG. 26).

The hepatocytes obtained in Example 1 were subcultured by another method. That is, after removing the medium from the petri dish, the colonies were treated with 0.02% EDTA and 0.05% trypsin, and the small hepatocytes and non-parenchymal cells of the colonies were dispersed in the solution. This cell dispersion was pipetted to obtain each dispersion of non-parenchymal cells around the colony and small hepatocytes (FIG. 27). Next, these dispersions were filtered with a filter having a pore size of 20 μm to remove aggregation of small cells and to substantially disperse individual cells (FIG. 28). When these cells were slightly seeded on a petri dish and cultured in a medium having the same composition as that used for the primary culture, both small hepatocytes and non-parenchymal cells adhered to the petri dish. In addition, division and proliferation of non-parenchymal cells were also observed. However, no proliferation and colony formation of small hepatocytes was observed (FIG. 29).

Therefore, in Example 1, the medium (conditio) used for the primary culture (1 to 4 days) for colony formation was used.
ned medium), good proliferation and colony formation of small hepatocytes were observed (FIG. 30).
This also suggests that the medium used for the primary culture of hepatocytes contains factors that activate the division and proliferation of small hepatocytes.

[0027]

As described above in detail, according to the present invention, a hepatic parenchymal cell having a clonal proliferative ability, which is thought to contain a hepatic progenitor cell, a method for obtaining the cell, and a method for inheriting the hepatic parenchymal cell Provided are methods for culturing in an alternative manner. This makes it possible to study in detail the development and differentiation processes of hepatocytes and the mechanisms of their growth and function development, and to elucidate the mechanisms of various human liver diseases, including liver cancer, and to develop therapeutic methods for them. A new way can be opened.

[Brief description of the drawings]

FIG. 1 is a phase-contrast microscope photograph (magnification 147) instead of a drawing showing the state of cells collected from 8-week-old rats on the third day of culture.

FIG. 2 is a phase contrast micrograph of the same visual field showing the state of the same cells as in FIG. 1 on day 5 of culture.

FIG. 3 is a phase-contrast microscope photograph of the same field of view showing the state of the same cells as in FIG. 1 on day 15 of culture.

FIG. 4 shows the relationship between the age of rats from which cells were collected and the number of hepatocyte colonies / cm ² on day 10 of culture.

FIG. 5 shows the relationship between the number of days of culture and the area of hepatocyte colonies for each rat age.

FIG. 6 is a micrograph (magnification: 75.8) instead of a drawing showing an HE-stained image of a hepatocyte colony on day 10 of culture of cells collected from an 8-week-old rat.

FIG. 7 shows culture 2 of cells collected from the same rat as in FIG.
It is a microscope picture (magnification 75.8) which showed the HE staining image of the hepatocyte colony on the 0th day.

FIG. 8 is a micrograph (magnification: 200) instead of a drawing showing a BD1-stained image of a hepatocyte colony on day 30 of culture of cells collected from a 7-week-old rat.

FIG. 9 is a micrograph (magnification: 152) replacing a drawing showing a cytokeratin 7-stained image of a hepatocyte colony on day 25 of culture of cells collected from an 8-week-old rat.

FIG. 10. Culture 25 of cells collected from 8-week-old rats
It is a microscope picture (magnification 606) which replaces a drawing and shows the albumin staining image of the hepatocyte colony on the day.

FIG. 11: Culture 25 of cells collected from 8-week-old rats
Alpha ₁ hepatocyte colonies in Japan th - is a microscopic photograph as a drawing which shows the anti-trypsin stained images (magnification 242).

FIG. 12. Culture of cells harvested from 7 week old rats 30
Hepatocyte colony transferrin-Br on day
Photomicrograph (magnification: 6) in place of a drawing showing a double stained image of dU.
06).

FIG. 13. Culture of cells harvested from 7 week old rats 30
It is a microscope photograph (magnification 152) substituted for a drawing which shows the alpha-fetoprotein staining image of the hepatocyte colony on the day.

FIG. 14. Culture of cells harvested from 7 week old rats 30
It is a microscope picture (magnification 152) substituted for the drawing which shows the GST-P staining image of the hepatocyte colony on the day.

FIG. 15. Culture of cells harvested from 7-week-old rats 30
15 is a micrograph (magnification: 152) instead of a drawing showing a γ-GTP-stained image of a hepatocyte colony on the day.

FIG. 16: Culture 2 of cells collected from 10-week-old rats
It is a microscope picture (magnification 152) substituted for the drawing which shows the OC2 staining image of the hepatocyte colony on the 2nd day.

FIG. 17: Culture 2 of cells collected from 10-week-old rats
It is a microscope picture (magnification: 152) instead of a drawing showing an OC3-stained image of a hepatocyte colony on the second day.

FIG. 18. Culture of cells harvested from 8-week old rats 25
It is a microscope picture (magnification 242) substituted for the drawing which shows the desmin staining image of the hepatocyte colony on the day.

FIG. 19 is a drawing which shows a phase contrast image of a hepatocyte colony on day 31 of culture when cells collected from an 8-week-old rat are cultured in a system in which all the additional factors are added to a medium (control). It is a microscope photograph (magnification 29.4).

FIG. 20 is a phase contrast micrograph of the same visual field when the same cells as in FIG. 19 are cultured in the EGF (−) system.

FIG. 21 is a phase contrast micrograph of the same field when the same cells as in FIG. 19 are cultured in a nicotinamide (−) system.

FIG. 22 is a phase contrast micrograph of the same field of view when the same cells as in FIG. 19 are cultured in an L-ascorbic acid phosphate (−) system.

FIG. 23 is a phase contrast micrograph of the same field of view when the same cells as in FIG. 19 are cultured in a DMSO (−) system.

FIG. 24 is a phase contrast micrograph of the same field when the same cells as in FIG. 19 are cultured in the FBS (−) system.

FIG. 25 is a transmission electron micrograph (a: 4250 ×, b: 21300 ×) of a hepatocyte colony on day 10 of culture of a cell colony collected from an 8-week-old rat.

FIG. 26 is a phase contrast micrograph (× 76) of a hepatic parenchymal cell subcultured by the method of the present invention on day 47 of culture.

FIG. 27 is a phase contrast micrograph (76) of the liver parenchymal cells dispersed by the method of the present invention and the non-parenchymal cells around the colony.
Times).

FIG. 28 is a phase contrast micrograph (× 76) of the cells in FIG. 27 after filtering the cells.

FIG. 29 is a phase contrast micrograph (30 ×) of small hepatocytes obtained by subculturing the cells of FIG. 28 with the same composition as the primary culture medium.
It is.

FIG. 30 is a phase contrast micrograph (30) of a small hepatocyte obtained by subculturing the cells of FIG. 28 in the medium used for primary culture itself.
Times).

Continuation of the front page (56) References JP-A-1-157377 (JP, A) JP-A-8-502175 (JP, A) Cell Structure and Function, 1989, Vol. 1, p. 75-Pathobiology, 1990, Vol. 58, No. 2, p. 65-77 Biochimi. Biophys. Acta. 1991, Vol. 1095, No. 2, p. 169-17 Experimental Cell Research, 1993, Vol. 2, p. 198 Virchows Archiv B cell Pathol, Vol. 5 (1992), p. 329-335 Proceedings of the Society for Experiential Biology and Medicine, Vol. 204, no. 3 (1993), p. 261-269 journal of Cellular Physiology, Vol. 151 (1992), p. 497-505 Tohoku J .; Exp. Med. , Vol. 132 (1980), p. 277- 287 (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 5/00-5/28 C12Q 1/04 BIOSIS (DIALOG) WPI (DIALOG)

Claims (8)

(57) [Claims] Claims 1. A hepatocyte is isolated from the liver of an adult mammal, and the hepatocyte is separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated from fetal bovine serum and A method for obtaining hepatic parenchymal cells having clonal proliferation ability, comprising culturing in a medium containing ascorbic acids as essential components to form colonies in hepatic parenchymal cells belonging to small cells. 2. Hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated from fetal bovine serum and Cultivation in a medium containing ascorbic acids as essential components causes colonization of hepatic parenchymal cells belonging to small cells, and then forms colonies with or without peroxisomes, reactivity to hepatocyte markers, reactivity to cancerous hepatocyte markers And a method for obtaining hepatic parenchymal cells having clonal proliferative ability, wherein screening is performed using at least one of the reactivity to an undifferentiated hepatocyte marker as an index. 3. The clonal expansion ability according to claim 2, wherein the small hepatocytes in the light fraction are cultured in a DMEM medium containing fetal calf serum, ascorbic acids, epidermal growth factor, nicotinamide and DMSO. A method for obtaining a parenchymal cell having liver. 4. The mammal according to claim 2, wherein the mammal is a rat.
The method for obtaining a hepatic parenchymal cell having a clonal proliferation ability according to any one of the above. 5. Hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated by centrifugation at a low speed into a heavy fraction and a light fraction, and the small hepatocytes in the light fraction are separated from fetal bovine serum and Primary culture in a medium containing ascorbic acids as essential to form colonies in hepatocytes belonging to small cells,
A method for subculturing hepatic parenchymal cells having clonal proliferation ability, comprising detaching colony cells from a medium with an EDTA solution and reculturing the detached cells in a medium having the same composition as the primary culture medium. . 6. A hepatocyte is isolated from the liver of an adult mammal, and the hepatocyte is separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated from fetal bovine serum and Primary culture in a medium containing ascorbic acids as essential to form colonies in hepatocytes belonging to small cells,
The clonality is characterized in that the colony cells are detached from the medium with an EDTA solution and a trypsin solution, the detached cells are dispersed in individual cells, and then recultured in the medium itself used at the beginning of the primary culture. A method for subculturing hepatocytes having the ability to proliferate. 7. The clonal expansion of claim 6 or 7, wherein the small hepatocytes in the light fraction are primarily cultured in a DMEM medium containing fetal calf serum, ascorbic acids, epidermal growth factor, nicotinamides and DMSO. A method for subculturing hepatocytes having ability. 8. The mammal according to claim 6, wherein the mammal is a rat.
The method for subculturing hepatic parenchymal cells having clonal proliferation ability of any one of the above.